Selective tyrosine kinase 2 (TYK2) inhibitor compounds, methods of synthesis, and uses

Abstract

This invention discloses a selective tyrosine kinase 2 (TYK2) inhibitor compound, its synthesis method, and its uses. The compound is shown in general formula (I). This compound exhibits certain TYK2 inhibitory activity in H9 cells. The compound effectively inhibits TYK2 activity. A time-dependent experiment was conducted after setting the dosage concentration. The optimal effect was observed at 24 hours with prolonged administration. Following this, a JAK kinase family selectivity experiment was performed, detecting the phosphorylation levels of downstream proteins in the JAK1 / JAK2 / TYK2 pathway, the JAK1 / JAK3 pathway, and the JAK2 pathway. The results showed that in the TYK2-free pathway, the phosphorylation level of downstream proteins remained unchanged or showed little change with increasing concentration. This indicates that compound B3 has high target selectivity, low off-target risk, and potentially high safety. This further suggests that the compound has potential for the treatment of psoriasis.

Description

Technical Field

[0001] This invention relates to compounds, their preparation methods and uses, and more specifically to selective tyrosine kinase 2 (TYK2) inhibitor compounds, their synthesis methods and uses. Background Technology

[0002] Psoriasis (PsO) is a chronic, relapsing, inflammatory skin disease mediated by the immune system. The suffering caused by this disease brings significant psychological and financial stress to patients. Immune system abnormalities are one of the main factors inducing psoriasis. Current psoriasis treatments mainly include biologics and small-molecule oral medications, with small-molecule inhibitors being the most widely used. Drug therapy can suppress psoriasis and improve patients' quality of life; however, current first-line treatments for moderate to severe psoriasis suffer from poor patient tolerance, poor selectivity, and drug resistance. Furthermore, the various side effects of small-molecule inhibitors cannot be ignored. Therefore, developing effective and safe psoriasis drugs is a major challenge for pharmaceutical researchers.

[0003] Traditional small-molecule inhibitors such as methotrexate, cyclosporine, and acitretin have serious drug interactions and long-term toxicity. Phosphodiesterase (PDE) inhibitors and Janus kinase (JAK) inhibitors play a crucial role in psoriasis treatment. Apemilast is the only clinically approved oral PDE-4 inhibitor for the treatment of psoriasis, treating moderate to severe psoriasis by reducing inflammation. Tofacitinib, baritinib, and ruxolitinib belong to the first-generation JAK inhibitors. Although their efficacy has been demonstrated to some extent, safety issues have resulted in a narrow therapeutic window and severe adverse reactions, including cardiotoxicity. Upadacitinib is a second-generation JAK inhibitor. Due to the same dose-dependent severe adverse reactions as first-generation JAK inhibitors, clinical trials related to psoriasis were terminated. Studies have shown that tofacitinib, utpatinib, and baricitinib do not inhibit TYK2 at therapeutic concentrations, which may indicate that the typical adverse reactions of JAK inhibitors are not caused by TYK2 inhibition. Therefore, the development of highly selective TYK2 inhibitors has attracted much attention from researchers. The selective TYK2 inhibitor deucravacitinib achieves an optimal balance between safety and efficacy. Currently, TYK2 has also become a popular target for the development of drugs for the treatment of psoriasis and other immune diseases.

[0004] Under both normal and pathological conditions, the JAK-STAT pathway plays a crucial role in intracellular signaling across various cellular processes. TYK2 influences the phosphorylation levels of STAT1 and STAT2 by regulating downstream signaling pathways of IL-12, IL-23, and type I IFN receptors. IL-23 and IL-17 are considered key cytokines for initiating and maintaining chronic inflammation, and TYK2 is a critical intracellular signal transduction link between them. Inhibiting TYK2 activity can sever the IL23 / IL-17 link, thereby inhibiting a key step in the pathogenesis of psoriasis. Blocking the TYK2-mediated JAK-STAT pathway has become a novel strategy for developing treatments for psoriasis.

[0005] Currently, the main treatments for psoriasis are biologics and small molecule drugs. However, biologics are expensive, and small molecule drugs, such as pan-JAK inhibitors, can cause serious adverse reactions. Deucravatinib, as the first approved TYK2 JH2 inhibitor, has achieved an optimal balance between selectivity and safety, bringing new hope to psoriasis patients. Therefore, the development of selective TYK2 inhibitors is of great significance. Summary of the Invention

[0006] Objective of the Invention: This invention provides a selective tyrosine kinase 2 (TYK2) inhibitor compound. Another objective of this invention is to provide a method for preparing the above-mentioned compound or a pharmaceutically acceptable salt thereof. A further objective of this invention is to provide the use of the said compound or a pharmaceutically acceptable salt thereof in the preparation of TYK2 inhibitors.

[0007] Technical solution: A compound as shown in general formula (I) or a pharmaceutically acceptable salt thereof:

[0008]

[0009] Wherein, R1 is selected from the following structures:

[0010]

[0011] R2 is a C1-C4 alkyl group or -H;

[0012] R3 is a C1-C4 alkyl group or

[0013] The compound or a pharmaceutically acceptable salt thereof,

[0014] R1 is selected from the following structure:

[0015]

[0016] R2 is -CH3 or -H;

[0017] R3 is -CH3, -C2H5, or...

[0018] The compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the following structures:

[0019]

[0020]

[0021]

[0022]

[0023] The method for preparing the compound or a pharmaceutically acceptable salt thereof includes the following steps:

[0024] Route 1:

[0025]

[0026] Among them, R1, R2, and R3 are the same as described above;

[0027] X and Y are selected from C or N, and the two are different;

[0028] R4: H, -CH3, -OCH3, F;

[0029] R5:

[0030]

[0031] or:

[0032] Route 2:

[0033]

[0034]

[0035] The preparation method described herein, wherein the reaction conditions in route 1 are as follows: (a) 4,6-dichloronicotinic acid, EDCI, HOBt, DIPEA, DCM, -5 to 5℃; (b) HATU, DIPEA, DMF, room temperature; (c) NH4Cl, Fe powder, EtOH, 80 to 90℃; (d) NaHMDS, THF; (e) cyclopropionamide, Cs2CO3, Xantphos, Pd(OAc)2, 1,4-dioxane, 130 to 150℃.

[0036] The preparation method described herein, wherein the reaction conditions in route 2 are as follows: (a) 4,6-dichloronicotinic acid, carbonyl diimidazole, methylamine hydrochloride, DIPEA, room temperature; (b) 3-nitro-2-(1H)-pyridone, copper acetate, pyridine, 1,4-dioxane, 70–90 °C; (c) NH4Cl, Fe powder, EtOH, 75–95 °C; (d) NaHMDS, THF; (e) cyclopropionamide, Cs2CO3, Xantphos, Pd(OAc)2, 1,4-dioxane, 130–150 °C.

[0037] A pharmaceutical composition comprising the said compound or a pharmaceutically acceptable salt thereof and pharmaceutically acceptable excipients.

[0038] The use of the compound or a pharmaceutically acceptable salt thereof in the preparation of TYK2 inhibitors.

[0039] The use of the compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating psoriasis.

[0040] The dosage forms of the drug include capsules, tablets, granules, pills, oral liquids, and injections.

[0041] This invention modifies the structure of the lead compound Deucravacitinib by replacing pyridazine with pyridine using the principle of skeletal migration and introducing groups on the amide side chain to increase the number of interaction sites with the target, resulting in a series of new compounds. These new compounds exhibit strong inhibitory activity and target selectivity, demonstrating good activity and safety, and are expected to serve as candidate compounds for further mechanistic exploration and in vivo experiments, providing a foundation for subsequent research on TYK2 JH2 inhibitors.

[0042] Beneficial Effects: Compared with existing technologies, compound B3 of this invention exhibits certain TYK2 inhibitory activity in H9 cells. Compound B3 effectively inhibits TYK2 activity at a concentration of 1 μM. A time-dependent experiment was then conducted with the dosage concentration set at 1 μM. The optimal effect was observed at 24 hours with prolonged administration. Following this, a JAK kinase family selectivity experiment was performed, detecting the phosphorylation levels of downstream proteins in the JAK1 / JAK2 / TYK2, JAK1 / JAK3, and JAK2 pathways. The results showed that in pathways lacking TYK2, the phosphorylation level of downstream proteins remained unchanged or showed little change with increasing concentration. This indicates that compound B3 possesses high target selectivity, low off-target risk, and potentially high safety. This further demonstrates the potential of this compound for the treatment of psoriasis. Attached Figure Description

[0043] Figure 1The results show the phosphorylation levels of STAT1 protein by 21 compounds, with Deucravacitinib as a positive control. Detailed Implementation

[0044] This invention provides a method for synthesizing selective TYK2 inhibitor compounds. The preparation method includes route 1 and route 2.

[0045] Route 1:

[0046]

[0047] Among them, R1, R2, and R3 are the same as described above;

[0048] X and Y are selected from C or N, and the two are different;

[0049] R4: H, -CH3, -OCH3, F;

[0050] R5:

[0051]

[0052] Reaction conditions: (a) 4,6-Dichloronicotinic acid, EDCI, HOBt, DIPEA, DCM, 0℃, 15min, 30%; (b) HATU, DIPEA, DMF, RT, overnight, 80%–90%; (c) NH4Cl, Fe powder, EtOH, 85℃, 1H, 95%; (d) NaHMDS, THF, 1H, 0℃, 25%; (e) Cyclopropionamide, Cs2CO3, Xantphos, Pd(OAc)2,1,4-dioxane, 145℃, 2h.

[0053] Route 2:

[0054]

[0055] Reaction conditions: (a) 4,6-Dichloronicotinic acid, carbonyl diimidazole, methylamine hydrochloride, DIPEA, RT, 4h, 90%–95%; (b) 3-nitro-2-(1H)-pyridone, copper acetate, pyridine, 1,4-dioxane, 80℃, overnight, 60%–70%; (c) NH4Cl, Fe powder, EtOH, 85℃, 1H, 95%; (d) NaHMDS, THF, 1H, 0℃, 25%; (e) Cyclopropionamide, Cs2CO3, Xantphos, Pd(OAc)2, 1,4-dioxane, 145℃, 2h.

[0056] Example 1: Synthesis of 4-[4-[[2-[cyclopropanecarbamoyl]-5-[methylcarbamoyl]pyridin-4-yl]amino]benzamido]piperidine-1-carboxylic acid tert-butyl ester (A1)

[0057] Synthesis route:

[0058]

[0059] Step 1:

[0060] 4,6-Dichloronicotinic acid (5 g) and carbonyl diimidazole (8.48 g) were dissolved in anhydrous THF (100 ml), and N2 was used for replacement. After stirring at room temperature for 30 min, methylamine hydrochloride (2.1 g) and N,N-diisopropylethylamine (DIPEA, 7.43 g) were added sequentially, and N2 was used for replacement. After reacting at room temperature for 4 h, the starting material disappeared as monitored by TLC. The N2 was removed, and the reaction was quenched by adding 60 mL of saturated NaHCO3 aqueous solution. Then, 300 mL of ethyl acetate was added for extraction. The extraction was repeated three times, and the upper organic phases were combined. The mixture was washed three times with saturated NaCl aqueous solution and finally dried with anhydrous Na2SO4. The solution was concentrated to obtain a white solid compound 1a (4.7 g), which did not require purification and was used directly in the next step. 1H NMR (300MHz, DMSO-d6) δ8.62 (s, 1H), 8.48 (s, 1H), 7.91 (s, 1H), 2.78 (d, J = 4.7Hz, 3H).

[0061] Step 2:

[0062] p-Nitrobenzoic acid (500 mg) and 4-amino-1-tert-butoxycarbonylpiperidine (863 mg) were dissolved in anhydrous DMF, and then HATU (1.71 g) and DIPEA (1.16 g) were added. The mixture was then dried in a drying tube and reacted overnight at room temperature. The reaction was monitored by TLC until the starting material disappeared. The reaction was quenched with water and extracted with ethyl acetate (50 mL × 3). The organic phases were combined and washed successively with saturated NaHCO3 (100 mL × 3) and NaCl (100 mL × 3). Finally, the mixture was dried with anhydrous Na2SO4, concentrated under reduced pressure, and then subjected to column chromatography to give a yellow oily compound 1d (900 mg, yield 86%). 1H NMR(300MHz,Chloroform-d)δ8.31–8.25(m,2H),8.14–8.08(m,2H),7.66(d,J=7.9Hz,1H),3.8 6(dp,J=7.8,4.6Hz,1H),3.59–3.49(m,4H),1.98–1.88(m,2H),1.77–1.67(m,2H),1.45(s,9H).

[0063] Step 3:

[0064] Compound 1d (890 mg) was dissolved in 10 mL of EtOH, followed by the addition of NH4Cl (1.4 g) aqueous solution and Fe powder (1.35 g) in sequence. The mixture was refluxed at 85 °C for 1 h, cooled to room temperature, filtered through diatomaceous earth, and the filter cake was washed with EtOH. The filtrate was concentrated under reduced pressure and purified by column chromatography to give a yellow oily compound 1e (770 mg, yield 94.8%). ¹H NMR (300 MHz, Chloroform-d) δ 7.64–7.52 (m, 2H), 6.70–6.58 (m, 2H), 5.29 (t, J = 3.1 Hz, 1H), 4.08 (s, 4H), 2.88 (t, J = 13.0 Hz, 2H), 1.98 (d, J = 12.4 Hz, 2H), 1.45 (q, J = 3.2 Hz, 9H).

[0065] Step 4:

[0066] Compound 1b (100 mg) and compound 1e (172 mg) were placed in a two-necked flask, 5 mL of anhydrous THF was added, and the mixture was replaced with N2. 1 M NaHMDS / THF solution (1.47 mL) was slowly added dropwise under ice bath conditions. The reaction was allowed to proceed for 1 h. TLC monitoring showed the disappearance of the starting material. N2 was removed, and the reaction was quenched with water. THF was removed by vacuum distillation, and the mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated NaCl aqueous solution, dried over anhydrous Na2SO4, and purified by vacuum distillation and column chromatography to give a white solid 1f (60 mg, yield 25.1%). mp:224℃-227℃.1H NMR(300MHz,Chloroform-d)δ10.32(d,J=3.1Hz,1H),8.36(d,J=3.3Hz,1H),7.78(dd,J=8.9,3.2Hz,2H),7.28–7.23(m,2H),7.06(d,J=3.3Hz,1H), 6.88(s,1H),6.20–6.07(m,1H),4.12(s,3H),3.02(d,J=4.1Hz,3H),2.98 –2.83(m,2H),2.03(d,J=12.5Hz,2H),1.81(s,2H),1.46(d,J=3.3Hz,9H).

[0067] Step 5:

[0068] Compound 1f (60 mg) was dissolved in 1,4-dioxane, and then cyclopropamide (31 mg) and Cs2CO3 (117 mg) were added sequentially. The mixture was substituted with N2 and stirred at room temperature for 15 min. Then Xantphos (10 mg) and Pd(OAc)2 (4 mg) were added and substituted with N2. The mixture was stirred at room temperature for 5 min and then heated to 145 °C and refluxed for 2 h. The reaction was monitored by TLC until the starting material disappeared. The N2 was removed, and the reaction was quenched with water. After the reaction solution was cooled to room temperature, it was filtered with diatomaceous earth. The filter cake was washed with methanol, and the filtrate was evaporated to dryness under reduced pressure. The solution was purified by column chromatography to obtain a white solid A1 (43 mg, yield 67.2%). 1HNMR(300MHz,DMSO-d6)δ10.80(s,1H),10.68(s,1H),8.65(d,J=4.8Hz,1H ),8.53(s,1H),8.24(d,J=7.8Hz,1H),8.12(s,1H),7.92–7.83(m,2H),7.30 (d,J=8.6Hz,2H),3.94(d,J=13.2Hz,3H),2.79(d,J=4.4Hz,5H),1.97(q,J= 6.1Hz,1H),1.79(d,J=12.0Hz,2H),1.41(s,11H),0.78(d,J=6.1Hz,4H).13C NMR(151MHz,DMSO-d6)δ173.42,168.33,165.23,154.50,154.41,151.73,149.21,142.29,129.6 6,129.27,120.65,109.75,95.97,79.09,46.94,40.53,28.57,26.42,14.78,8.26.HRMS(ESI)m / z calcd forC28H36N6O5[M+H]+536.2720; found 537.2816.HPLC (70% methanol in water): tR=10.593min, 96.2%.

[0069] Example 2: Synthesis of 4-(3-((2-(cyclopropanecarbamoyl)-5-(methylaminocarbamoyl)pyridin-4-yl)amino)benzoyl)piperidine-1-carboxylic acid tert-butyl ester (A2)

[0070] Following the synthesis method of Example 1, p-nitrobenzoic acid was replaced with nitrobenzoic acid, while other conditions remained unchanged. 1H NMR (400MHz, DMSO-d6) δ10.77(s,1H),10.58(s,1H),8.63(q,J=4.5Hz,1H),8.52(s,1H),8.1 8(d,J=7.9Hz,1H),7.95(s,1H),7.69(t,J=2.0Hz,1H),7.59(dt,J=7.5,1.6Hz,1H),7.50–7.4 0(m,2H),4.12(q,J=5.7,4.8Hz,1H),4.07–3.87(m,4H),3.17(d,J=3.0Hz,2H),2.78(d,J=4.4 Hz,3H),1.97(p,J=6.3Hz,1H),1.77(dd,J=13.2,3.9Hz,2H),1.40(s,9H),0.86–0.74(m,4H).

[0071] Example 3: Synthesis of 4-(3-((2-(cyclopropanecarbamoyl)-5-(methylaminocarbamoyl)pyridin-4-yl)amino)-2-methoxybenzamido)piperidine-1-carboxylic acid tert-butyl ester (A3)

[0072] Following the synthesis method of Example 1, p-nitrobenzoic acid was replaced with 2-methoxy-3-nitrobenzoic acid, while other conditions remained unchanged. 1 H NMR(300MHz,Chloroform-d)δ10.51(s,1H),9.09(s,1H),8.34(s,1H),8.07(s,1H),7 .86–7.74(m,2H),7.63(dd,J=8.0,1.7Hz,1H),7.33–7.28(m,1H),6.79(d,J=5.1Hz,1H ),4.28–4.05(m,3H),3.83(s,3H),3.06(d,J=4.7Hz,3H),2.98(d,J=12.5Hz,2H),2.1 0–1.99(m,2H),1.63(dt,J=8.0,3.5Hz,1H),1.10(p,J=4.3Hz,2H),0.96–0.81(m,4H). 13C NMR(151MHz,DMSO-d6)δ173.34,168.36,165.42,154.51,154.34,152.23,149.59,149.07,133.29,131.68,124.53,124 .35,123.45,109.88,95.34,79.10,61.88,55.37,46.57,31.60,28.55,26.41,14.76,13.69,8.21,6.66.HRMS(ESI)m / z calcd for C 29 H 39 N6O6[M+H] + 567.3926; found 567.2917. HPLC (70% methanol in water): tR=12.810min, 95.1%.

[0073] Example 4: Synthesis of 4-(3-((2-(cyclopropanecarbamoyl)-5-(methylaminocarbamoyl)pyridin-4-yl)amino)-3-methoxybenzamido)piperidine-1-carboxylic acid tert-butyl ester (A4)

[0074] Following the synthesis method of Example 1, p-nitrobenzoic acid was replaced with 3-methoxy-4-nitrobenzoic acid, while other conditions remained unchanged. mp: 232℃-234℃. 1H NMR (300MHz, DMSO-d6) δ10.81(s,1H),10.63(s,1H),8.62(d,J=4.8Hz,1H),8.53(s,1H),8.25(d,J=7.8Hz,1H),8.14(s,1H),7.57–7. 43(m,3H),3.98(d,J=11.6Hz,3H),3.92(s,3H),2.87(s,2H),2.80(d,J=4.3Hz,3H),2.00(q,J=6.0Hz,1H),1.81(d,J=11.6Hz,2H),1.4 5(m,2H),1.43(s,9H),0.80(d,J=6.1Hz,4H).13CNMR(151MHz,DMSO-d6)δ173.40,168.18,165.24,154.45,154.38,151.29,150.37,14 9.10,131.49,129.78,120.35,118.62,111.02,110.35,96.19,79.10,56.41,47.02,40.54,28.57,26.45,14.78,8.26.HRMS(ESI)m / z calcd for C29H39N6O6[M+H]+567.3926; found567.2914.HPLC (70% methanol in water): tR=11.647min, 98.4%.

[0075] Example 5: Synthesis of 4-(6-((2-(cyclopropanecarbamoyl)-5-methylaminocarbamoyl)pyridin-4-yl)amino)nicotinamide)piperidine-1-carboxylic acid tert-butyl ester (A5)

[0076] Following the synthesis method of Example 1, p-nitrobenzoic acid was replaced with 6-aminonicotinic acid, while other conditions remained unchanged. mp: 240℃-244℃. 1H NMR (300MHz, DMSO-d6) δ 11.64 (s, 1H), 10.81 (s, 1H), 9.30 (s, 1H), 8.76 (d, J = 4.7Hz, 1H), 8.60 (s, 1H), 8.54 (d, J = 7.7Hz, 1H), 8.41 (d, J = 5.2Hz, 1H), 7.41 (s, 1H), 7.34 (dd, J = 5.3, 1.3Hz, 1H), 3.96 (d, J = 13.7Hz, 3H), 2.83 (d, J = 4.4Hz, 3H), 2.04 (s, 1H), 1.82 (d, J = 12.5Hz, 3H). Hz,2H),1.43(s,9H),1.25(s,2H),0.84(t,J=5.7Hz,4H).13CNMR(151MHz,DMSO-d6)δ173.04,168.51,164.08,154.66,154.45,154. 37,150.01,148.85,148.63,144.00,115.27,111.16,109.84,99.72,79.13,47.16,40.54,28.56,26.53,14.82,8.21.HRMS(ESI)m / z calcd for C27H35N7O5[M+H]+538.2772; found 538.2764.HPLC (70% methanol in water): tR=9.995min, 95.7%.

[0077] Example 6: Synthesis of 4-(4-((2-((cyclopropanecarbamoyl)-5-methylcarbamoyl)pyridin-4-yl)amino)-3-methylbenzamido)piperidine-1-carboxylic acid tert-butyl ester (A6)

[0078] Following the synthesis method of Example 1, p-nitrobenzoic acid was replaced with 3-methyl-4-nitrobenzoic acid, while other conditions remained unchanged. mp: 187℃-190℃. 1H NMR(300MHz,Chloroform-d)δ10.25(s,1H),8.56(s,1H),8.27(s,1H),7.89(s,1H) ,7.67(d,J=2.1Hz,1H),7.53(dd,J=8.4,2.1Hz,1H),7.41(d,J=8.3Hz,1H),6.53(s ,1H),6.20(d,J=7.8Hz,1H),2.99(d,J=4.7Hz,3H),2.89(t,J=12.6Hz,2H),2.33(s ,3H),1.97(s,3H),1.47(s,9H),1.05–1.00(m,2H),0.85(dq,J=7.7,4.0Hz,4H).13C NMR(151MHz,DMSO-d6)δ173.31,168.55,165.41,154.45,154.40,152.48,149.14,140.64,130.68,130.58,1 30.34,126.35,121.25,109.26,95.70,79.08,55.37,46.93,28.56,26.44,18.15,14.74,8.21.HRMS(ESI)m / z calcd for C29H39N6O5[M+H]+551.2976; found 551.2962.HPLC (70% methanol in water): tR=12.445min, 96.5%.

[0079] Example 7: Synthesis of 4-(4-((2-((cyclopropanecarbamoyl)-5-(methylcarbamoyl)pyridin-4-yl)amino)-2-methylbenzamido)piperidine-1-carboxylic acid tert-butyl ester (A7)

[0080] Following the synthesis method of Example 1, p-nitrobenzoic acid was replaced with 2-methyl-3-nitrobenzoic acid, while other conditions remained unchanged. mp: 120℃-122℃. 1H NMR(300MHz,Chloroform-d)δ10.08(s,1H),8.47(s,1H),8.21(s,1H),7.35(s, 1H),6.34(d,J=4.9Hz,1H),6.21(d,J=8.3Hz,1H),4.08(t,J=6.6Hz,2H),2.99( d,J=4.7Hz,3H),2.92–2.77(m,2H),2.27(s,3H),2.00(d,J=12.0Hz,2H),1.62( dt,J=19.9,7.1Hz,2H),1.46(s,9H),1.37–1.31(m,1H),0.95–0.84(m,4H).13C NMR(151MHz,DMSO-d6)δ173.15,169.70,168.66,168.63,154.39,154.35,153.59,149.07,140.06,138.36,129.30,126.70,124.74,123.99,1 08.75,94.99,79.09,64.97,64.23,55.37,46.56,43.52,30.54,30.45 ,28.54,26.43,19.03,14.78,14.69,13.99,13.97,8.14.HRMS(ESI)m / z calcd for C29H39N6O5[M+H]+551.2976; found 551.5959.HPLC (70% methanol in water): tR=9.595min, 97.5%.

[0081] Example 8: Synthesis of 4-(5-((2-(cyclopropanecarbamoyl)-5-(methylcarbamoyl)pyridin-4-yl)amino)pyridinecarbamoyl)piperidine-1-carboxylic acid tert-butyl ester (A8)

[0082] Following the synthesis method of Example 1, p-nitrobenzoic acid was replaced with 5-aminonicotinic acid, while other conditions remained unchanged. mp: 156℃-160℃. 1H NMR (300MHz, Chloroform-d) δ 10.58 (s, 1H), 8.46 (d, J = 2.5Hz, 2H), 8.33 (s, 1H), 8.23–8.11 (m, 2H), 7.85 (d, J = 8.3Hz, 1H), 7.74 (dd, J = 8.5, 2.6Hz, 1H), 6.50 (d, J = 5.2Hz, 1H) ),4.26–3.85(m,3H),3.01(d,J=4.7Hz,3H),2.92(d,J=12.7Hz,2H),1.52(s,2H),1.47 (s,9H),1.26(d,J=8.1Hz,1H),1.07(p,J=4.3Hz,2H),0.88(dq,J=7.6,4.1Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.53,168.09,163.17,154.60,154.38,151.42,149.30,145.17,141.46,138.9 4,129.26,123.26,110.26,96.04,79.10,55.38,46.83,31.64,28.57,26.44,14.78,8.32.HRMS(ESI)m / z calcd for C27H35N7O5[M+H]+538.2772; found538.2768.HPLC (70% methanol in water): tR=12.408min, 98.6%.

[0083] Example 9: Synthesis of 4-(4-((2-((cyclopropanecarbamoyl)-5-methylaminocarbamoyl)pyridin-4-yl)amino)-3-fluorobenzamido)piperidine-1-carboxylic acid tert-butyl ester (A9)

[0084] Following the synthesis method of Example 1, p-nitrobenzoic acid was replaced with 3-fluoro-4-nitrobenzoic acid, while other conditions remained unchanged. mp: 234℃-238℃. 1H NMR(300MHz,DMSO-d6)δ10.86(d,J=14.8Hz,2H),8.72(d,J=4.8Hz,1H),8.59(s,1 H),8.35(d,J=7.7Hz,1H),8.04(s,1H),7.86–7.72(m,2H),7.61(t,J=8.2Hz,1H),3 .96(d,J=12.6Hz,3H),2.85(s,2H),2.81(d,J=4.4Hz,3H),2.00(q,J=6.1Hz,1H), 1.81(d,J=12.4Hz,2H),1.43(s,9H),1.42–1.22(m,2H),0.81(d,J=6.1Hz,4H).13C NMR(151MHz,DMSO-d6)δ173.51,168.26,164.12,154.95,154.59,154.40,153.34,151.42,149.13,130.81,130.77,130.43 ,130.35,124.49,124.47,121.67,115.58,115.44,109.91,96.21,79.09,47.10,28.55,26.43,14.79,8.31.HRMS(ESI)m / z calcd forC28H36FN6O5[M+H]+555.2726; found 555.2718. HPLC (70% methanol in water): tR=12.635min, 98.2%.

[0085] Example 10: Synthesis of 4-(4-((2-((cyclopropanecarbamoyl)-5-methylaminocarbamoyl)pyridin-4-yl)amino)-4-fluorobenzamido)piperidine-1-carboxylic acid tert-butyl ester (A10)

[0086] Following the synthesis method of Example 1, p-nitrobenzoic acid was replaced with 4-fluoro-3-nitrobenzoic acid, with other conditions remaining unchanged. mp: 248℃-252℃. 1H NMR (300MHz, DMSO-d6) δ 10.81 (s, 1H), 10.55 (s, 1H), 8.68 (d, J = 4.6Hz, 1H), 8.56 (s, 1H), 8.13 (d, J = 7.8Hz, 1H), 8.04–7.95 (m, 1H), 7.73 (d, J = 14.7Hz, 2H), 7.45 (t, J = 9.5Hz, 1H),3.97(s,2H),3.92(s,1H),2.88(d,J=13.4Hz,2H),2.81(d,J=4.3Hz,3H),1.98(m, 1H),1.79(d,J=12.5Hz,2H),1.42(s,9H),0.89(t,J=7.2Hz,2H),0.81–0.74(m,4H).13C NMR (151MHz, DMSO-d6) δ173.37,169.70,168.32,164.56,158.24,156.58,154.47,154.29,152.54,148.98,131.92,127.04,126. 96,125.86,125.80,123.93,116.80,109.37,95.85,79.06,47.02,30.54,28.54,26.41,19.02,14.75,13.99,8.18.HRMS(ESI)m / z calcd for C28H36FN6O5[M+H]+555.2726; found 555.2717. HPLC (70% methanolin water): tR=15.340min, 97.7%.

[0087] Example 11: Synthesis of 6-(cyclopropaneformamido)-4-((2-methoxy-3-(morpholino-4-carbonyl)phenyl)amino)-N-methylnicotinamide (B1)

[0088] Following the synthesis method of Example 3, 4-amino-1-tert-butoxycarbonylpiperidine was replaced with morpholine, while other conditions remained unchanged. mp: 158℃-161℃. 1H NMR (300MHz, Chloroform-d) δ 10.27 (s, 1H), 9.04 (s, 1H), 8.22 (s, 1H), 7.84 (s, 1H), 7.43 (dd, J = 8.0, 1.6Hz, 1H), 7.15 (t, J = 7.8Hz, 1H), 7.02 (dd, J = 7.6, 1.6Hz, 1H), 6.91 (d, J = 5.3H). z,1H),3.75(d,J=8.8Hz,7H),3.69–3.48(m,2H),3.42–3.19(m,2H),2.93(d,J=4.6Hz,3H) ,1.56(dq,J=7.9,4.5,4.0Hz,1H),0.99(p,J=4.2Hz,2H),0.82(dq,J=7.5,4.1Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.28,168.37,166.63,154.44,152.38,148.98,148.80,132.92,132.11,132.04,131.28,125.04 ,123.58,123.40,109.68,95.59,66.72,66.48,61.66,55.37,47.58,42.13,26.42,14.74,8.19,8.16.HRMS(ESI)m / zcalcd for C23H27N5O5[M+H]+454.2085; found 454.2077.HPLC(75%methanol inwater):tR=3.902min,97.7%.

[0089] Example 12: Synthesis of 6-(cyclopropaneformamido)-4-((3-(dibutylcarbamoyl)-2-methoxyphenyl)amino)-N-methylnicotinamide (B2)

[0090] Following the synthesis method of Example 3, 4-amino-1-tert-butoxycarbonylpiperidine was replaced with di-n-butylamine, while other conditions remained unchanged. mp: 174℃-179℃. 1H NMR(300MHz,Chloroform-d)δ10.36(s,1H),8.39(s,1H),8.27(s,1H),8.07(s ,1H),7.53(dd,J=8.1,1.6Hz,1H),7.21(t,J=7.8Hz,1H),7.01(dd,J=7.6,1.6H z,1H),6.42(d,J=5.2Hz,1H),3.84(s,3H),3.16(dd,J=30.9,11.7Hz,4H),3.0 4(d,J=4.7Hz,3H),1.67(d,J=7.6Hz,1H),1.54–1.41(m,4H),1.19–1.07(m,4H) ,1.01(t,J=7.3Hz,3H),0.90(td,J=6.9,6.0,3.6Hz,4H),0.80(t,J=7.3Hz,3H ).13CNMR(151MHz,DMSO-d6)δ173.25,168.39,167.87,154.51,152.38,149.01 ,148.17,133.03,124.95,122.83,122.77,109.70,95.51,61.64,47.97,43.70 ,30.45,29.48,26.41,20.02,19.58,14.73,14.28,13.87,8.17.HRMS(ESI)m / z calcd for C27H38N5O4[M+H]+496.2918; found498.2910.HPLC (75% methanol in water): tR=13.332min, 99.1%.

[0091] Example 13: Synthesis of 4-((3-(butylcarbamoyl)-2-methoxyphenyl)amino)-6-(cyclopropanecarbamoyl)-N-methylnicotinamide (B3)

[0092] Following the synthesis method of Example 3, 4-amino-1-tert-butoxycarbonylpiperidine was replaced with n-butylamine, while other conditions remained unchanged. mp: 164℃-168℃. 1H NMR (300MHz, DMSO-d6) δ 10.80 (s, 1H), 10.67 (s, 1H), 8.64 (q, J = 4.4Hz, 1H), 8.52 (s, 1H), 8.28 (t, J = 5.7Hz, 1H), 8.04 (s, 1H), 7.54–7.51 (m, 1H), 7.22–7.19 (m, 1H), 6.78 (s... ,1H),3.70(s,3H),3.26(q,J=6.6Hz,2H),2.79(d,J=4.4Hz,3H),2.05–1.91(m,1H),1 .51(td,J=5.0,2.2Hz,2H),0.91(t,J=7.2Hz,3H),0.77(s,2H),0.63–0.60(m,4H).13C NMR(151MHz,DMSO-d6)δ175.12,173.34,168.37,165.94,154.52,152.24,149.66,149.07,133.29,131.6 6,124.55,123.46,109.88,95.35,61.88,39.12,31.59,26.41,20.08,14.16,13.69,6.66.HRMS(ESI)m / z calcd for C23H29N5O4[M+H]+440.2292; found 440.2283.HPLC (75% methanol in water): tR=5.852min, 97.6%.

[0093] Example 14: Synthesis of 6-(cyclopropaneformamido)-4-((2-methoxy-3-((tetrahydro-2H-pyran-4-yl)carbamoyl)phenyl)amino)-N-methylnicotinamide (B4)

[0094] Following the synthesis method of Example 3, 4-amino-1-tert-butoxycarbonylpiperidine was replaced with 4-aminotetrahydropyran hydrochloride, while other conditions remained unchanged. mp: 138℃-140℃. 1H NMR (300MHz, Chloroform-d) δ 10.43 (s, 1H), 9.80 (s, 1H), 8.28 (s, 1H), 7.99 (s, 1H), 7.77 (d, J = 7.8Hz, 1H), 7.65 (dd, J = 7.9, 1.6Hz, 1H), 7.52 (dd, J = 8.0, 1.6Hz, 1H), 7.22–7.08 (m, 2H), 4.17–4.09 (m, 1H), 3.97 (dt) ,J=11.9,3.6Hz,2H),3.78(s,3H),3.51(td,J=11.5,2.2Hz,2H),2.98(d,J=4.5Hz,3H),1.93(d,J=4.0Hz, 1H),1.61–1.50(m,2H),1.24(t,J=7.1Hz,2H),1.06(p,J=4.1Hz,2H),0.86(dq,J=7.6,4.5,4.1Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.35,168.38,165.39,154.52,152.25,149.61,149.08,133.30,131.66,124.5 2,124.37,123.46,109.90,95.35,66.40,61.87,60.22,45.81,32.70,26.41,14.77,8.21.HRMS(ESI)m / z calcd for C24H30N5O5[M+H]+468.2242; found 468.2234.HPLC (75% methanol in water): tR=4.145min, 99.1%.

[0095] Example 15: Synthesis of 6-[cyclopropaneformamido]-4-[[2-methoxy-3-[piperidin-4-carbamoyl]phenyl]amino]-N-methylnicotinamide (B5)

[0096]

[0097] Step 1:

[0098] A3 (100 mg) was dissolved in 3 mL of ethyl acetate, and 0.1 mL of 36% HCl was added. The mixture was reacted at room temperature for 1 h. The starting material disappeared as monitored by TLC. The pH was adjusted to 7-8 with saturated NaHCO3 solution, and the mixture was concentrated under reduced pressure and purified by column chromatography to give a white solid B5 (78 mg, 95%). mp: 236℃-238℃. 1H NMR (300MHz, DMSO-d6) δ11.05(s,1H),9.18(d,J=4.8Hz,1H),9.06(s,1H),8.84(d,J=9.9Hz ,1H),8.58(d,J=7.5Hz,1H),8.51(s,1H),7.53(dd,J=7.8,1.8Hz,1H),7.35(dd,J=7.7,1.8 Hz,1H),7.30–7.23(m,2H),4.06(s,1H),3.70(s,3H),3.32(s,2H),3.05–2.95(m,2H),2.81 (d,J=4.4Hz,3H),2.00(ddt,J=10.2,7.3,4.4Hz,3H),1.86–1.73(m,2H),1.00–0.90(m,4H). 13 C NMR(151MHz,DMSO-d6)δ165.78,150.87,132.25,130.94,124.82,110.52,9 4.84,62.12,55.40,44.54,42.29,28.31,26.52,15.23,9.50.HRMS(ESI)m / z calcd for C 24 H 30 N6O4[M+H] + 467.2401; found 467.2395.

[0099] Example 16: Synthesis of 6-(cyclopropaneformamido)-4-((2-methoxy-3-(4-methylpiperazin-1-carbonyl)phenyl)amino)-N-methylnicotinamide (B6)

[0100] Following the synthesis method of Example 1, p-nitrobenzoic acid was replaced with (3-amino-2-methoxyphenyl)(4-methyl-1-piperazinyl) methyl ketone, with other conditions remaining unchanged. mp: 226℃-228℃. 1H NMR (300MHz, DMSO-d6) δ 10.78 (s, 1H), 10.60 (s, 1H), 8.65 (d, J = 4.7Hz, 1H), 8.53 (s, 1H), 7.96 (d, J = 7.6Hz, 1H), 7.46 (dd, J = 8.1, 1.6Hz, 1H), 7.21 (t, J = 7.8Hz, 1H), 6.99 (dd, J = 7.6, 1.5Hz, 1H). 1H),3.67(s,3H),3.20(d,J=5.0Hz,2H),3.07(q,J=7.3Hz,2H),2.80(d,J=4.3Hz,3H),2.48–2 .27(m,4H),2.24(s,3H),2.04–1.94(m,1H),1.20(t,J=7.3Hz,2H),0.78(d,J=4.8Hz,2H).13C NMR(151MHz,DMSO-d6)δ173.27,168.37,166.46,154.46,152.31,149.00,148.65,132.95,131.60,12 4.96,123.19,123.14,109.72,95.59,61.61,55.13,46.04,41.41,26.43,14.73,8.16.HRMS(ESI)m / z calcd for C24H30N6O4[M+H]+467.2401; found 467.2395.HPLC (75% methanol in water): tR=4.067min, 97.1%.

[0101] Example 17: Synthesis of 4-((3-(butylcarbamoyl)-2-methoxyphenyl)amino)-6-(cyclopropanecarbamoyl)-N-ethylnicotinamide (C1)

[0102] Following the synthesis method of Example 1, methylamine hydrochloride was replaced with ethylamine hydrochloride, while other conditions remained unchanged. mp: 142℃-148℃. 1H NMR(300MHz,Chloroform-d)δ10.49(s,1H),9.61(s,1H),8.31(s,1H),8.10(s ,1H),7.82(dd,J=16.9,6.8Hz,2H),7.62(d,J=7.9Hz,1H),7.35–7.25(m,1H), 6.82(t,J=5.5Hz,1H),3.82(s,3H),3.50(dq,J=13.9,6.6Hz,4H),1.72(dt,J= 7.9,3.6Hz,1H),1.63(t,J=7.5Hz,2H),1.48(tt,J=8.2,4.9Hz,4H),1.31(t,J =7.2Hz,3H),1.08(p,J=4.2Hz,2H),0.90(dq,J=6.4,3.5Hz,2H),0.81(dt,J=7 .3,3.7Hz,4H).13CNMR(151MHz,DMSO-d6)δ175.11,173.35,167.71,165.93,1 54.52,152.27,149.63,133.31,131.64,124.54,123.40,110.02,95.34,61.8 7,39.12,34.27,31.60,20.08,15.08,14.16,13.69,8.21,6.66.HRMS(ESI)m / z calcd for C24H31N5O4[M+H]+454.2449; found454.2449.HPLC (75% methanol in water): tR=6.767min, 99.2%.

[0103] Example 18: Synthesis of 4-((3-(butylcarbamoyl)-2-methoxyphenyl)amino)-6-(cyclopropanecarbamoyl)-N-cyclopropylnicotinamide (C2)

[0104] Following the synthesis method of Example 1, methylamine hydrochloride was replaced with cyclopropylamine, while other conditions remained unchanged. ¹H NMR (300 MHz, Chloroform-d) δ 10.44 (s, ¹H), 8.77 (s, ¹H), 8.23 ​​(s, ¹H), 8.04 (s, ¹H), 7.84–7.71 (m, 2H), 7.59 (dd, J = 8.0, 1.7 Hz, ¹H), 7.27 (s, ¹H), 6.64 (s, ¹H), 3.79 (s, 3H), 3.52– 3.40(m,2H),2.90(dq,J=7.2,3.6Hz,1H),1.60(s,1H),1.42(dt,J=8.0,4.6Hz,4H),1.07 –1.01(m,2H),0.98(d,J=2.7Hz,3H),0.91–0.88(m,2H),0.79(dd,J=5.8,2.2Hz,4H).13C NMR(151MHz,DMSO-d6)δ175.12,173.36,169.28,165.94,154.56,152.16,149.63,149.31,133.28,131.65,124 .54,123.38,109.77,95.33,61.90,39.13,31.59,23.26,20.08,14.16,13.69,8.22,6.66,6.16.HRMS(ESI)m / z calcd for C25H32N5O4[M+H]+466.2449; found466.2453.HPLC (75% methanol in water): tR=6.990min, 98.2%.

[0105] Example 19: Synthesis of 4-((3-(butylcarbamoyl)-2-methoxyphenyl)amino)-6-(cyclopropanecarbamoyl)-N,N-dimethylnicotinamide (C3)

[0106] Following the synthesis method of Example 1, methylamine hydrochloride was replaced with dimethylamine, while other conditions remained unchanged. mp: 158℃-162℃. 1H NMR (300MHz, Chloroform-d) δ 9.50 (s, 1H), 8.85 (s, 1H), 8.18 (s, 1H), 8.06 (s, 1H), 7.84–7.72 (m, 2H), 7.62 (dd, J = 8.0, 1.7Hz, 1H), 7.28 (t, J = 7.5Hz, 1H), 3.81 (s, 3H), 3.48 (td, J = 7.5Hz, 1H). .0,5.6Hz,2H),3.18(s,6H),1.62–1.57(m,1H),1.47–1.41(m,2H),1.07(dt,J=6.5,3.4Hz ,2H),0.98(d,J=3.8Hz,4H),0.87(dq,J=7.4,3.9Hz,2H),0.79(dt,J=8.0,3.4Hz,3H).13C NMR (151MHz, DMSO-d6) δ175.10,173.25,168.25,165.88,153.72,150.70,149.68,148.33,133.41,131.36,124. 53,124.47,123.65,113.42,96.85,61.83,39.13,31.60,20.08,14.73,14.17,13.69,8.14,6.66.HRMS(ESI)m / z calcd for C24H31N5O4[M+H]+454.2449; found 454.2446.HPLC(75% methanol in water): tR=5.140min, 94.7%.

[0107] Table 1. Other compounds of the present invention

[0108]

[0109]

[0110]

[0111] Example 20: Synthesis of 6-(cyclopropaneformamido)-4-((1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridin-3-yl)amino)-N-methylnicotinamide (A11)

[0112] Synthesis route:

[0113]

[0114] Step 1:

[0115] The synthesis method is the same as step 1 of Example 1.

[0116] Step 2:

[0117] 3-Nitro-2-(1H)-pyridone (1g), 4-fluorophenylboronic acid (1.5g), copper acetate (2.13g), and pyridine (8.6mL) were added sequentially to 24mL of 1,4-dioxane and reacted overnight at 80°C. The reaction was quenched with water, the solvent was evaporated under reduced pressure, and the mixture was extracted with ethyl acetate (80mL×3). The organic phases were combined and washed sequentially with 10% HCl aqueous solution and saturated NaCl aqueous solution. The mixture was dried over anhydrous Na2SO4 and the solvent was evaporated under reduced pressure to obtain a white solid 11d, which was directly added to the feed.

[0118] Step 3:

[0119] The synthesis method was the same as step 3 of Example 1, yielding a white solid 11e (299 mg, yield 67.3%). 1 H NMR(300MHz,Chloroform-d)δ7.45–7.32(m,2H),7.16(t,J=8.6Hz,2H),6.75(dd,J =6.9, 1.7Hz, 1H), 6.58 (dd, J = 7.2, 1.7Hz, 1H), 6.14 (t, J = 7.0Hz, 1H), 4.30 (s, 2H).

[0120] Step 4:

[0121] The synthesis method was the same as step 4 of Example 1, yielding a light yellow solid compound 11f (274 mg, 50.2%). 1 H NMR(300MHz,Chloroform-d)δ9.80(s,1H),8.76(s,1H),7.76(dd,J=7.3,1.3Hz,1H),7.51(q,J=5 .1Hz,1H),7.48–7.42(m,2H),7.16–7.08(m,3H),7.00(s,1H),6.82(s,1H),2.91(d,J=4.9Hz,3H).

[0122] Step 5

[0123] The synthesis method was the same as step 5 of Example 1, yielding a white solid compound A11 (168 mg, 54%). mp: 260℃-268℃. 1H NMR(300MHz,DMSO-d6)δ10.88(s,1H),10.73(s,1H),8.56(q,J=4.5Hz,1H), 8.51(s,1H),8.25(s,1H),7.60–7.48(m,2H),7.45(dd,J=7.3,1.6Hz,1H),7. 41–7.33(m,2H),7.31(dd,J=7.0,1.6Hz,1H),6.41(t,J=7.1Hz,1H),2.76(d, J=4.3Hz, 3H), 2.03 (tt, J=7.6, 3.7Hz, 1H), 0.83 (tq, J=8.4, 5.6, 4.2Hz, 4H). 13 C NMR(75MHz,DMSO-d6)δ173.61,167.70,163.53,160.28,158.04,154.56,150.26,149.03,137.48,137.44,131.19, 130.93,129.50,129.38,119.23,116.50,116.20,111.37,105.87,96.98,26.37,14.86,8.36.HRMS(ESI)m / zcalcd for C 22 H 21 FN5O3[M+H] + 422.1623; found 422.1616. HPLC (75% methanol in water): tR=4.698min, 97.7%.

[0124] Example 21: Synthesis of A12

[0125] Following the synthesis method of Example 20, 4-fluorophenylboronic acid was replaced with phenylboronic acid, while other conditions remained unchanged. 1 HNMR(300MHz,DMSO-d6)δ10.88(s,1H),10.73(s,1H),8.54(d,J=18.7Hz,2H),8.25(s,1H),7.61–7.50(m,2H),7.50–7.41(m, 4H),7.32(dd,J=6.9,1.6Hz,1H),6.41(t,J=7.1Hz,1H),2.76(d,J=4.4Hz,3H),2.03(h,J=5.5,5.0Hz,1H),0.90–0.76(m,4H). 13C NMR(75MHz,DMSO-d6)δ173.61,167.70,157.95,154.56,150.28,149.03,141.26,131.20,130.94, 129.57,128.79,127.18,119.20,111.37,105.84,96.97,26.37,14.86,8.35.HRMS(ESI)m / zcalcd for C 22 H 22 N5O3[M+H] + 404.1717; found 404.1713. HPLC (75% methanol in water): tR=4.690min, 98.4%.

[0126] Table 2. Other compounds of the present invention

[0127]

[0128] Example 22: Activity Test

[0129] Table 3 lists the sources of the instruments used in the following activity tests.

[0130] Table 3. Source of Instruments

[0131]

[0132]

[0133] (I) Cell Counting Kit-8 (CCK-8) assay for the cell safety of compounds

[0134] 1. Experimental Methods: H9 cells were cultured in RPMI-1640 complete medium (containing 10% FBS) at 37°C with 5% CO2. When the cell density reached 80%-90%, half-volume medium replacement was used for passage. 5 mL of fresh medium was added to a T25 culture flask, and after thorough mixing, half of the cell suspension was transferred to a new culture flask for further culture. This half-volume medium replacement method was repeated 2-3 times. Afterward, the cell suspension was transferred to centrifuge tubes and centrifuged at 1000 rpm for 5 min. The supernatant was discarded to remove dead cells and repeatedly used medium. Finally, fresh medium was added, and the suspension was thoroughly mixed with a pipette. Equal volumes of cell suspension were then transferred to two separate culture flasks and cultured at 37°C with 5% CO2. When the cell density reached 80%, cells in the logarithmic growth phase were used to prepare a solution with a concentration of 6 × 10⁻⁶ cells / mL. 4Cell suspension at 100 μL / mL was seeded into 96-well plates and incubated for 4 h. A pre-prepared 10 mM DMSO stock solution was diluted with culture medium to 100 μM, 40 μM, and 20 μM, respectively. 100 μL of the diluted solution was then added to each well of the 96-well plate to achieve final drug concentrations of 50 μM, 20 μM, and 10 μM, with three replicates for each concentration. Replicas containing the same volume of culture medium instead of the drug-containing medium served as the control group, while replicates containing neither cells nor drug-containing culture medium served as the blank group. After 24 h of incubation, 20 μL of CCK-8 reagent was added to each well, and the plates were incubated in the dark for 4 h. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%

[0135] 2. Experimental Results:

[0136]

[0137]

[0138] Using Deucravacitinib as a positive control, the safety of the synthesized nicotinamide compounds in CCK8 cells was tested. The results showed that most compounds were safe for H9 cells. At a concentration of 10 μM, compounds A1, A4, A8, and A9 significantly inhibited H9 cell growth. The cell survival rate of the remaining compounds was greater than 80%, which was better than that of the positive control drug Deucravacitinib. At 50 μM, most compounds caused the survival rate of H9 cells to be less than 50%. Therefore, 10 μM was selected as the administration concentration for the subsequent phosphorylated STAT1 protein content test.

[0139] (II) Western Blot assay to detect the phosphorylation level of STAT1 protein

[0140] 1. Experimental Method:

[0141] (1) Protein extraction

[0142] Logarithmically growing H9 cells were prepared into a suspension of 8 × 10⁶ cells / mL using RPMI-1640 medium and seeded into 6-well plates, with 1 mL of cell suspension in each well. After culturing for 4 h, 1 mL of 20 μM diluent of the test compound was added, bringing the final concentration of the compound in each well to 10 μM. One hour after drug administration, 5 μL of 20 μg / mL IFN-α was added to stimulate the cells. After 30 min, the cell suspension in the 6-well plate was transferred to 2 mL of IFN-α solution. Centrifuge at 1000 rpm for 5 min at 4 °C in EP tubes, discard the supernatant, add 1 mL of 4 °C PBS to each EP tube to wash cells, centrifuge at 1000 rpm for 5 min at 4 °C, discard the supernatant, add 50 μL of pre-prepared RIPA lysis buffer (RIPA:PMSF:phosphorylase inhibitor = 100:1:2) to each tube to lyse the cells, lyse on ice for 30 min, vortex once every 15 min, after lysis, centrifuge at 12000 rpm for 15 min at 4 °C, take the supernatant and use BCA kit to quantify protein, measure absorbance at 562 nm, calculate the loading volume required for 30 μg of protein, add loading buffer and denature the protein, store in a -80 °C freezer.

[0143] (2) Making adhesive

[0144] Using a 10% SDS-PAGE gel, first prepare the separating gel (lower gel solution: lower gel buffer: coagulant = 1 mL: 1 mL: 20 μL), mix well, pour into the gel casting plate, add an appropriate amount of methanol or water, flatten the separating gel, and after the lower gel solidifies (15 min to 30 min), discard the upper methanol or water, and absorb excess methanol or water with filter paper; then prepare the upper gel (lower gel solution: lower gel buffer: coagulant = 1 mL: 1 mL: 20 μL), mix well, pour into the gel casting plate, slowly insert the comb, and after solidification, place in Tris-Glycine electrophoresis buffer, remove the comb, and prepare for sample loading.

[0145] (3) Sample loading-electrophoresis

[0146] Add the prepared protein sample to the well of the precast gel according to the calculated loading volume. After concentrating at 60V for 40 minutes, observe the marker at 120V constant voltage to see if the protein has separated. Usually, stop electrophoresis after the bromophenol blue has moved to the bottom of the gel.

[0147] (4) Transfer membrane

[0148] After electrophoresis, cut the corresponding target protein gel band in the transfer buffer, place the gel on transfer filter paper, cut the PVDF membrane of the corresponding size, activate it in methanol for 10 min, place it on the corresponding gel band and remove air bubbles, then cover it with transfer filter paper and sponge, and finally place it in the transfer clamp for transfer at a constant current of 200mA and in an ice bath. The transfer is completed after 70 min.

[0149] (5) Closed

[0150] After the transfer is complete, the PVDF membrane is transferred to an antibody incubation box, washed three times with TBST solution, and then 5 mL of 5% skim milk or 5% BSA solution is added to block non-specific protein sites on the membrane at room temperature for 1 hour.

[0151] (6) Antibody incubation and development

[0152] After blocking, the blocking solution was aspirated and the bands were washed three times with TBST. Then, diluted primary antibody was added and incubated at 4°C for 12-16 hours. After the incubation, the primary antibody was recovered and the bands were washed three times with TBST for 5 minutes each time. Finally, a suitable concentration of secondary antibody dilution was added and the bands were incubated at room temperature for 2 hours. The secondary antibody dilution was aspirated and the bands were washed three times with TBST for 5 minutes each time. Finally, the bands were developed using a chemiluminescence imaging system.

[0153] 2. Experimental Results

[0154] like Figure 1 As shown, using Deucravacitinib as a positive control, the phosphorylation level of STAT1 protein by 21 compounds was detected. Compound B3 was selected as a candidate compound, and concentration- and time-dependent experiments and JAK family selectivity experiments were conducted.

[0155] (III) Concentration and time-dependent experiments of compound B3

[0156] 1. Experimental Methods

[0157] (1) Concentration-dependent experiment

[0158] Log-grown H9 cells were prepared into 8×10⁸ cells using RPMI-1640 medium. 6 A suspension of cells / mL was seeded into 6-well plates, with 1 mL of cell suspension seeded into each well. After incubation for 4 h, 1 mL of 20 μM, 10 μM, 2 μM, and 1 μM diluent of the test compound was added to each well, resulting in final concentrations of 10 μM, 5 μM, 1 μM, and 0.5 μM for each compound. One h after drug administration, 5 μL of 20 μg / mL IFN-α was added to stimulate the cells, and protein was extracted after 30 min.

[0159] (2) Time-dependent experiment

[0160] H9 cells were prepared into 8×10⁸ cells using RPMI-1640 medium. 6 Cells were seeded with a suspension of 1 mL / mL into 6-well plates. After incubation for 4 h, 1 mL of 2 μM diluent of the test compound was added to each well, resulting in a final concentration of 1 μM in each well. Cells were stimulated with 5 μL of 20 μg / mL IFN-α after 24 h, 10 h, 6 h, 2 h, and 1 h of drug administration. Proteins were extracted after 30 min.

[0161] 2. Experimental Results

[0162] Four dosing concentrations were designed in total. Compound B3 significantly inhibited the JAK1 / TYK2 pathway at a concentration of 1 μM, and the inhibitory effect of compound B3 increased with increasing concentration. Based on the concentration-dependent experiment results, the time-dependent experiment was conducted at a dosing concentration of 1 μM, with a total of 5 dosing times. After reaching the corresponding dosing time, cells were stimulated with IFN-α, and proteins were extracted for Western blotting experiments 30 min later. The inhibitory effects on the signaling pathway were similar between 1 and 10 h, and the inhibitory effect was still present at 24 h, with better results than other dosing times.

[0163] B3 exhibits good TYK2 inhibitory activity. Cellular safety assays using a CCK-8 assay kit, Western blotting analysis of STAT1 protein phosphorylation, and concentration- and time-dependent assays of compound B3 indicate that the 21 compounds described above can serve as selective TYK2 inhibitors for the treatment of autoimmune diseases, particularly psoriasis.

Claims

1. A compound of Formula (I) or a pharmaceutically acceptable salt thereof: Formula (I). The compound is selected from the following structures:

2. A process for the preparation of a compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, comprising the following steps: Scheme 1: wherein R1, R2, R3 are as defined in claim 1; X, Y are C; R4 is -OCH3; R5is selected from 3. The preparation method according to claim 2, characterized in that, The reaction conditions in Scheme 1 are: (a) 4,6-dichloronicotinic acid, EDCI, HOBt, DIPEA, DCM, -5-5 °C; (b) HATU, DIPEA, DMF, room temperature; (c) NH4Cl, Fe powder, EtOH, 80-90 °C; (d) NaHMDS, THF; (e) cyclopropanecarboxamide, Cs2CO3, Xantphos, Pd(OAc)2, 1,4-dioxane, 130-150 °C.

4. A pharmaceutical composition, characterized by, comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

5. Use of a compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a TYK2 inhibitor.

6. Use of a compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating psoriasis.

7. Use according to claim 6, characterized in that, The dosage form of the medicament includes capsules, tablets, granules, pills, oral liquids, injections.

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